Genetic and biochemical analysis of Col and R plasmids with particular emphasis on the plasmids ColE1, R6K, RK2 and mini-F and in vitro generated derivatives of ColEl carrying unrelated specified regions of prokaryotic DNA. Temperature-sensitive mutants defective in ColEl replication will be analyzed at a non-permissive temperature for defects in relaxation complex, formation of RNA-containing plasmid DNA and the generation of replicative intermediates. Attempts will be made to determine the origin and directionality of replication of various plasmids, including joint replicons, and the role of RNA priming in the synthesis of these elements. An electron microscopy analysis of plasmid DNA-membrane complexes will be carried out to probe the role of membranes in plasmid DNA replication. The role of relaxation complex in the replication and conjugal transfer of plasmids also will be emphasized. Finally, reconstitution of plasmids will be carried out in vitro to construct plasmids that are more useful as cloning vehicles. BIBLIOGRAPHIC REFERENCES: Plasmid ColEl DNA Replication in Escherichia coli Strains Temperature-sensitive for DNA Replication. John Collins, Peter Williams and Donald R. Helinski. Molec. Gen. Genet. 136:273-289 (1975). Bidirectional Replication of Plasmid R6K DNA in Escherichia coli; correspondence between origin of replication and position of single-strand break in relaxed complex. Michael A. Lovett, Robert B. Sparks and Donald R. Helinski. Proc. Nat. Acad. Sci. USA 72:2905-2909 (1975).